Method of treatment of amyotrophic lateral sclerosis with a protein extractable from mammalian organs

ABSTRACT

A method of treatment of patients affected by amyotrophic lateral sclerosis comprising the administration of an effective amount of a 14 kDa protein extractable from mammalian organs, particularly mammalian liver.

[0001] The present invention concerns a method of treatment of patientsaffected by amyotrophic lateral sclerosis comprising the administrationof an effective amount of a 14 kDa protein extractable from mammalianorgans, particularly mammalian liver.

[0002] Amyotrophic lateral sclerosis (incidence of 1.4 to 4.7/100.000)also named motor neuron disease, is a degenerative disease characterizedby progressive paralysis which affects elderly subjects (65-70 years),developing into complete paralysis and death and in a short time.

[0003] Several biochemical and genetic factors seem to be involved inthe pathogenesis of ALS, which remains however to be still elucidated.An increase in some intracellular proteins (cytoskeleton) which affectcell activity and neurotransmission, seems to be the main cause ofamyotrophic lateral sclerosis.

[0004] Although the aetiology of ALS is still largely unknown, accordingto a recent hypothesis, the first step in the onset of ALS appears to beconnected with an increase of toxic factors such as oxygen radicals andthe cited formation of protein cytoskeleton whereas the degenerative andprogressive phase would seem to be at least partially activated andsustained by autoimmune mechanisms.

[0005] There is not specific treatment at present.

[0006] No experimental therapy proposed until now seemed particularlypromising.

[0007] One of the major difficulties in developing an effectivetreatment for ALS is due to the lack of a reliable and predictive animalmodel so that the only definitive evidence on the actual effectivenessof a new therapy has to be obtained from clinical tests.

[0008] It has now been found that ALS can be effectively treated byadministering to affected patients a 14 kDa protein which is normallypresent in mammalian liver, particularly in goat liver, and which can beprepared either by extraction or by recombinant DNA methods.

[0009] Said protein, hereinafter referred to with the abbreviation ofMFP 14 (derived from Multiple Function Protein 14 kDa) has beendisclosed by Ceciliani et al., FEBS Lett., 1996;393;147-50.

[0010] Its cytotoxic activity has been reported in Int.J.Oncol., 1996;8:543-48 whereas its cDNA and expression in E.coli is reported byColombo et al. in Biochem. Biophys. Acta, 1998;1442:49-59.

[0011] The preparation of the extractive protein as well as thepreparation of the recombinant protein have been respectively disclosedin U.S. Pat. No. 5,792,744 and in PCT/EP/00 03003 which are hereinincorporated by reference.

[0012] Therapeutic uses of this protein in the treatment of AIDS,autoimmune disease and TNF-induced disease have been disclosed in WO98/42366.

[0013] Moreover, said protein has been found to be an inhibitor ofprotein synthesis, a modulator of cytokines synthesis as well asspecific calpain activator.

[0014] MFP 14 has some sequence similarities with Heat shock proteins orHSP, with the protein binding to the Major Histocompatibilty Complex-1(MHC-1 binding protein) and with the YER057C/YIL051C/Y5GF family ofproteins having a still unknown function, highly evolutionary conservedfrom prokaryotes to mammals.

[0015] The invention, according to a first embodiment, providestherefore a method of treatment of ALS comprising the administration topatients in need of such treatment of a therapeutically active dose ofMFP 14 or active fragment.

[0016] The invention also provides pharmaceutical compositions usefulfor treating amyotrophic lateral sclerosis containing as the activecomponent an MFP 14 protein or active fragment.

[0017] The term MFP 14 refers also to proteins having high degree ofhomology with the amino acid sequence disclosed in the above-citedreferences. By high degree of homology, proteins having at least 70%homology with the 137 amino acid sequence of the native protein aremeant. Preferably, the degree of homology is higher than 80%, morepreferably higher than 90%.

[0018] The term “active fragment” refers to shorter sequences derivedfrom the native or recombinant MFP 14 protein and still retaining thepharmacological activity of the parent sequence. It is in fact knownthat the therapeutic activity of a given protein does not always requirea complete sequence, the activity being often confined to smallerregions, e.g. to N-terminal, Carboxy-terminal or internal regions. Insuch an event, it may be advantageous the administration of the activefragment rather than the intact protein in view of lower productioncosts, higher metabolic stability and other possible advantagesconnected with the administration of polypeptides having lower molecularweight.

[0019] The fragments and homologues of MFP 14 may also derive fromdeletion, substitutions and/or insertion mutation of amino acids. Forinstance, it is known that the so called “conservative” mutations, i.e.the substitution of an amino acid with another one of the same category(acidic, basic, neutral, hydrophilic or lipophilic), is usuallyacceptable for the preservation of activity.

[0020] The use of recombinant MFP 14 is particularly preferred in viewof the easier availability and standardization of production methods.

[0021] Alternatively, an extract comprising MFP 14 such as thatdisclosed in WO 92/10197 may also be used.

[0022] For the considered therapeutic use, MFP 14 or active fragmentsthereof will be administered parenterally, e.g. by intramuscular orsubcutaneous route, in form of sterile solutions or suspensions inacceptable carriers such as saline solutions, oils for parenteraladministration and the like.

[0023] Other administration routes can also be envisaged, for instancethe oral or transdermal route, using known methods for the delivery ofproteins or polypeptides by these routes (e.g. by means of liposomes ormicro-encapsulation methods).

[0024] The administration of MFP 14 proteins could also be carried outusing gene therapy protocols, for instance by administering suitablevectors, which may deliver to target cells a gene sequence coding forMFP 14. Suitable vectors as well as corresponding control sequences andprotocols are disclosed in FASEB J. 9, 190-199, 1995 and in Nature 392(suppl. 30 April ) 25-30, 1998.

[0025] MFP 14 dose range which was found to be effective in thetreatment of ALS is comprised from about 1 mg to 10 mg/day.

[0026] The dose can be divided in more than one daily administration,for instance two or three administrations. In particular cases, theadministrations can also be separated one from the other by longerperiod of times, up to 1-4 weeks. This can particularly apply to thechronic long-term treatment, once the first cycle of treatment has beencompleted.

[0027] The dosage regimen can anyhow vary within wide limits, in view ofthe very low toxicity of MFP 14, so that the skilled physicians willeasily adapt the doses according to individual patients' requirements,particularly taking into consideration the age, sex, weight of thepatient and the seriousness and advancement stage of the disease.

[0028] It has also been found that the administration of ubiquitin incombination with MFP 14 is advantageous in the treatment of ALS.Ubiquitins belong to a well-known family of proteins, the use of whichhas been proposed for several pathologies, which do not have anything incommon with ALS. For the considered therapeutic use, ubiquitins will beadministered, preferably contemporaneously, together with MFP 14, at adosage ranging from about 1 mg to 10 mg/day.

[0029] According to a further embodiment, the invention providestherefore also pharmaceutical compositions comprising as the activeingredient a combination of MFP 14 and of ubiquitin, in admixture with asuitable pharmaceutical carrier.

[0030] The administration of MFP 14 or of fragments thereof, optionallyin combination with ubiquitin, proved to be effective in clinical trialscarried out on patients with ALS at different stages. In particular thetreatment of the invention turned out to be effective both in the firststages as well as in the late stages of this pathology, inducing asignificant recovery of the motion function and an improvement of thesocial life in affected patients.

[0031] The following examples are given to further illustrate theinvention in more detail.

EXAMPLE 1

[0032] Pharmaceutical Composition of MFP 14 in Form of Vials forParenteral Administration Lyophilised Recombinant MFP 14 mg 0.5 obtainedaccording to PCT/EP/00 03003 Sterile Saline Solution ml 2

EXAMPLE 2

[0033] Pharmaceutical Composition of MFP14 and Ubiquitin in Form ofVials for Parenteral Administration Lyophilised Recombinant MFP 14 mg0.5 obtained according to PCT/EP/00 03003 Ubiquitin mg 1 Sterile SalineSolution ml 2

EXAMPLE 3

[0034] Clinical Tests

[0035] Four patients suffering from amyotrophic lateral sclerosis for2/4 years were treated.

[0036] In three patients, the first and second motor neurons wereaffected, with marked atrophy at the distal extremities of the upperlimbs and spasticity of the lower limbs. In the fourth patient, alsodementia was present (ALS plus dementia). In all patients, phonation anddeglutition were remarkably impaired, the fourth patient was fed throughnasal-gastric probe.

[0037] After about 15 days in the first three patients and already after7 days in the patient with dementia-ALS a marked improvement ofdeglutition and phonation was observed. Limb symptoms remained steady inthree patients; in one patient an improvement in strength anddeambulation was observed.

[0038] After one-month treatment, the results remained steady and theworsening typical of the disease was not observed even after severalweeks.

1. A method of treatment of patients affected by amyotrophic lateralsclerosis comprising the administration to patients in need of suchtreatment of a therapeutically active dose of MFP 14 or active fragmentthereof.
 2. A method according to claim 1 wherein MFP 14 is selectedfrom recombinant MFP 14 or MFP extracted from goat liver.
 3. A methodaccording to claim 1 further comprising the administration of Ubiquitin.